The molecular process of viral fusion, in which viral coat proteins recognize and bind to surface receptors of the host cell, is a critical target in the prevention and treatment of viral infections. Upon recognition of the viral glycoprotein by host cellular receptors, viral fusion proteins undergo conformational changes that are essential to viral fusion and infection. A series of hydrophobic amino acids, located at the N- and C-termini organize to form a complex that pierces the host cell membrane. Adjacent viral glycoproteins containing two amphipathic heptad repeat domains fold back on each other to form a trimer of hairpins, consisting of a bundle of six α-helices. This six-helix bundle motif is highly conserved among many viral families, including Filovirus (ebola), (Malashkevich, V. N., et al., PNAS, 1999. 96(6): p. 2662-2667; Weissenhorn, W., et al., Molecular Cell, 1998. 2(5): p. 605-616), Orthomyxovirus (influenza) (Wilson, I. A., J. J. Skehel, and D. C. Wiley, Nature, 1981. 289(5796): p. 366-37; Bullough, P. A., et al., Nature, 1994. 371(6492): p. 37-43), Coronavirus (SARS) (Xu, Y. H., et al. Journal of Biological Chemistry, 2004. 279(47): p. 49414-49419), Paramyxovirus (HRSV) (Zhao, X., et al., PNAS, 2000. 97(26): p. 14172-14177) and Retrovirus (HIV) (Chan, D. C., et al., Cell, 1997. 89(2): p. 263-27; Weissenhorn, W., et al., Nature, 1997. 387(6631): p. 426-430).
HIV envelope proteins gp120 and gp41 non-covalently associate with each other to form a trimer of dimers. On the host cell, gp120 specifically interacts with CD4, CXCR4, and CCR5, which are the glycoproteins involved in host-cell recognition. gp41, the viral membrane spanning glycoprotein, is responsible for fusing the viral and cellular membranes, resulting in viral particle uptake by the host cell. Once gp120 binds to CD4, gp41 undergoes a conformational change, transforming from its native state into a fusogenic six-helix bundle. The regions of gp41 involved in this change are 43 (C43) residues of the C-terminal heptad repeat (CHR or HR-2), near the transmembrane domain, and 51 (N51) residues of the N-terminal heptad repeat (NHR or HR-1), found just proximal to the fusion peptide domain. Peptides N51 and C43 orient to form helical antiparallel heterodimers, which associate to form a higher order trimeric complex that is thermo- and proteolytically stable.
Peptides which interfere with this viral fusogenic process can be used for the prevention and treatment of viral infections. For example, peptides corresponding to residues 553-590 of the gp41 N-terminal heptad repeat domain (HR-1) and residues 630-659 and 648-673 of the C-terminal heptad repeat domain (HR-2) of HIV have been shown to inhibit the replication of a variety of HIV strains. Studies have determined that these peptides inhibit cell-cell fusion by interacting with the HIV envelope glycoproteins.
T20 or enfuvirtide, is the first fusion inhibitor peptide developed based on the CHR region of gp41 for the treatment of HIV. Enfuvirtide is active at nanomolar concentrations against many strains and subtypes of HIV, including the common lab strains and primary isolates of HIV-1 and HIV-2 (Wild, C. T., et al., PNAS, 1994. 91(21): p. 9770-9774).
However, enfuvirtide has remained a tertiary treatment option due to a variety of factors which include cost, no oral bioavailability (subcutaneous injections limit accessibility and compliance) and poor in vivo stability (Kilby, J. M., et al., Nucleic Aids Research and Human Retroviruses, 2002. 18(10): p. 685-693), and loss of bioactive secondary structure. Thus, although peptide-based inhibition of viral fusion processes is mechanistically feasible and clinically effective, the biophysical and biochemical properties of amphipathic fusion peptides present numerous challenges which hinder their use.